We have continued studies of the distribution of the histone variants H3.3 and H2A.Z in chicken erythroid cells. We had shown recently that H3.3 is concentrated at many regulatory regions, as well as over the transcribed regions of some active genes. We then showed that nucleosomes containing H3.3 are less stable than those containing the predominant species, H3.1, and lose their H2AH2B components more readily. Furthermore, nucleosomes containing H3.3 and H2A.Z are even more unstable. Using double chromatin immunoprecipitation and gentle isolation methods we have identified numerous genomic loci at where such doubly substituted nucleosomes are present. They mark transcriptional regulatory sites that control active genes. In collaboration with the laboratory of Dr. Keji Zhao, NHLBI, we have now completed a genome-wide study of the distribution of nucleosomes containing both variants, together and separately. Double variant nucleosomes are highly concentrated at transcription start sites of transcriptionally active genes, and to a lesser extent at their transcription termination sites. They are also found over Dnase hypersensitive sites that typically mark regulatory elements. Single-variant nucleosomes are distributed quite differently, probably reflecting different functions. Our results allow us to present a novel picture of promoters of active genes, in which unstable nucleosomes and transcription factors share occupancy through rapid exchange. We have now developed methods for reconstructing single nuceleosome core particles containing various combinations of bacterially expressed histone variants, and an analytical ultracentrifuge technique for measuring their stability. This will enable us to determine whether it is the secondary structure of the histones, or variant-specific histone covalent modifications that is responsible for stability differences.